Project Summary/Abstract Improved understanding of stem cell biology has resulted in widespread investigation of the therapeutic potential of mesenchymal stem cells (MSCs) for restoration of tissue/organ integrity. However, poor survival rate of MSCs refutes the hypothesis that the donor cells can functionally integrate with the damaged tissue to facilitate the repair or regeneration process. Instead, mounting evidence corroborates that the paracrine mechanisms mediated by factors released by the MSCs play a critical role in the reparative process. Therefore, harnessing MSC secretome seems a reasonable approach for rational design of MSC based therapies. Despite the importance of cell-extracellular matrix (ECM) interactions in stem cell fate and the centrality of physico-mechanical as well as biochemical properties of the extracellular environment in the regulatory functions of ECM, the interplay of these factors or their combined roles in regulating the paracrine signaling of the stem cells has not been demonstrated. Therefore, the goal of the proposed work is to elucidate/optimize the synergies between different microenvironmental factors in guiding the pro-angiogenic signaling of MSCs. Our preliminary studies indicated that culture of MSCs on decellularzied ECM (dECM) enhanced their angiogenic activity. Also, our studies have suggested that MSCs prefer matrix of optimal stiffness for maximal release of vascular endothelial growth factor (VEGF). Based on these observations, we hypothesize that the mechanical and biochemical properties of ECM synergistically regulate MSC secretome. In this study, the composition of the matrices deposited by MSCs from different donors will be characterized as a function of media supplementation and oxygen tension (Aim 1). A dECM-polyethylene glycol (PEG) hybrid scaffold, the physico-chemical properties and functional activity of which can be manipulated independently, will be developed to assess the synergy of different mechano-chemical factors in fostering the pro-angiogenic signaling of MSCs (Aim 2).